Extended half-life and elevated steady-state level of a single-chain Fv intrabody are critical for specific intracellular retargeting of its antigen, caspase-7

Abstract Two single-chain Fv (sFv) antibodies (C8 and H2) specific for Mch3/caspase-7, a component in the signaling pathway for induction of apoptosis, were genetically fused to different intracellular targeting signals and analyzed by expression in mammalian cells. Immunofluorescence microscopy confirmed that these anti-caspase-7 intrabodies were expressed in the cellular compartments dictated by their C-terminal trafficking signals. Cytosolic caspase-7 was successfully retargeted to different subcellular compartments by specific intrabodies through direct association of antigen with intrabody. Sequestration of caspase-7 in nuclei had a significant biological impact in that the expression of a nuclear-targeted anti-caspase-7 intrabody in a stable Jurkat cell line markedly inhibited staurosporine-induced apoptosis. The criteria for choosing an optimal intrabody were also evaluated in this study. A gene dosage titration study demonstrated that the C8 intrabody was more potent in retargeting of caspase-7 than the H2 intrabody, even though the H2 sFv had a higher affinity for caspase-7 than the C8. Pulse-chase experiments and western blot analysis revealed that the anti-caspase-7 C8 sFv intrabodies exhibited a long half-life (>8 h) and high steady-state levels of protein accumulation, while the H2 intrabodies had a half-life of 2 h and less protein at steady state. These results suggest that the choice of sFv as an intrabody depends critically on the intracellular sFv protein having an extended half-life and elevated steady-state level. Thus, extended half-life must be considered together with sFv antibody specificity and affinity when choosing an optimal sFv intrabody for functional studies of cellular proteins.